Project Summary/Abstract Immune effector mechanisms that confer protection against HIV acquisition remain poorly understood. Immune-correlates analysis of the Phase III RV144 trial of the prime/boost ALVAC+gp120 protein vaccine, which delivered an overall 31.2% reduction in HIV acquisition, suggested a protective potential in anti-gp120 antibody responses. Specifically, of the 6 primary immunologic parameters evaluated in the RV144 case- control study, high IgG responses to the V1V2 loop of HIV envelope (Env) gp120 significantly correlated with a reduced risk of HIV acquisition. This correlate of protection has since been recapitulated in the SIV/macaque model. Moreover, in a subset of vaccinees with lower levels of neutralizing antibodies and Env-specific plasma IgA, antibody responses to the V3 loop and antibodies mediating antibody-dependent cellular cytotoxicity (ADCC) were also associated with protection. However, it remains unclear if and how these V1V2- and V3- specific antibodies directly mediate their protective effects. These antibodies have no broad neutralizing activity, but target conserved regions of the V1V2 and V3 loops and are capable of mediating Fc-dependent antiviral functions in vitro. Nonneutralizing antibodies against conserved conformational epitopes also synergize to enhance ADCC activity. We propose herein to investigate the protective potential and mechanisms of action of antibodies targeting V1V2, V3, and other, similarly immunogenic, conserved Env epitopes by testing passively administered human monoclonal antibodies (mAbs) in a humanized mouse (hu- mice) model. The proposed study is based on our preliminary findings that a nonneutralizing V1V2 mAb and a weakly neutralizing V3 mAb were each able to reduce virus infection rate and/or virus burden in hu-mice that were engrafted with CD34+ human hematopoietic stem cells and challenged with a tier 2 JRFL HIV-1 virus. The extent to which these and other anti-Env antibodies inducible by vaccination confer protection against diverse HIV-1 strains has not been determined, and their antiviral mechanisms also have not been defined. Therefore, we propose experiments to, first, evaluate the ability of anti-V1V2 and anti-V3 human mAbs to protect against virus infection in hu-mice upon challenge with HIV-1 isolates, particularly transmitted/founder viruses from subtypes B and C. Second, we propose testing vaccine-induced human mAbs against conserved epitopes in the V3 and constant regions of Env in hu-mice, using the same mAb transfer/virus challenge approach. Third, we will investigate the Fab and Fc contributions of these mAbs in protection against HIV. To this end, we will measure the mAb capacity to target free virions and infected cells, including ex vivo virions and cells from virus-infected hu-mice, in vitro. We will also prepare mAbs with Fc mutations that abrogate or enhance Fc-receptor interactions and test their ability to protect hu-mice against virus challenge. Data from this proposed study will help us understand the types of antibodies and immunogenic Env epitopes that, due to their protective potential, should be considered in future HIV vaccine development.